This application relates to the field of cancer imaging and therapy using novel somatostatin analogs.
Cancer is a leading cause of morbidity and mortality in developed countries. For example, approximately 1.4 million new cases of cancer and more than 0.5 million cancer deaths were reported in the U.S. in 1996. In 1995, the total annual cost of cancer care in the U.S., including direct and indirect costs, was estimated to be more than $96 billion. A great need exists for improved diagnostic and therapeutic tools to allow early detection and safe, cost-effective treatment of cancer.
Many tumors express receptors for the peptide hormone somatostatin. In particular, neuroendocrine tumors such as pituitary adenomas, pheochromocytomas, paragangliomas, some medullary thyroid carcinomas, and some small cell lung cancers express somatostatin receptors (SSTRs). In addition, cells of nervous system tumors such as astrocytomas and meningiomas display SSTRs on their surfaces. SSTR expression has also been found in human breast tumors, malignant lymphomas, and renal cell carcinomas, and some prostate tumors may be characterized by SSTR expression.
Binding studies performed with radiolabeled or iodinated somatostatin and its analogs have identified five SSTR subtypes (SSTRs1-5). The SSTR-bearing tumors described above express SSTR2 and SSTR5 most frequently, with SSTR3 and SSTR4 occurring less frequently, according to most authors. One group reports that SSTR3 is expressed at very high levels in almost all human tumors (Virgolini (1997) Eur. J. Clin. Invest. 27, 793-800). There is general agreement that most tumors typically express more than one SSTR subtype, and that varying densities of SSTRs may be expressed in the cells contained within a particular tumor. The availability of cloned SSTR subtype genes has allowed somatostatin analogs to be characterized by their affinities for SSTR1-5, and these studies have revealed considerable variability in SSTR subtype specificity among somatostatin analogs, as described in Raynor, et al. (1993) Molecular Pharmacol. 43, 838-844 and in Patel, et al. (1997) TEM 8, 398-404.
Until recently, three somatostatin analogs have been commercially available. Octreotide (Sandostatin(copyright)) binds to SSTR2, SSTR3, and SSTR5, and is marketed in the U.S. and Europe for treatment of acromegaly and control of symptoms associated with vipomas and metastatic carcinoid tumors. Lanreotide (Somatuline(trademark)) has a SSTR subtype profile similar to that of octreotide and is approved in several European countries for the same indications as octreotide. A radiolabeled form of octreotide, 111In-pentetreotide (111In-DTPA-D-Phe1-octreotide or 111In-OctreoScan(copyright)) has been approved in the U.S. and Europe for imaging neuroendocrine tumors.
Recently the U.S. Food and Drug Administration approved a new radiopharmaceutical, NeoTect(trademark), a 99mTc-labeled form of the novel somatostatin analog depreotide (P829), for sale as an imaging agent. Blum, et al., (1999) Chest 115, 224-232, describes the use of 99mTc-depreotide for evaluation of solitary pulmonary nodules of the lung. 99mTc-labeled depreotide has also been studied as an imaging agent for other somatostatin-receptor bearing tumors. For example, Handmaker (1998) J. Nucl. Med. 39, 315P describes a comparison of 99mTc-depreotide and 99mTc-sestamibi for diagnosis of breast cancer. Depreotide is described in commonly assigned U.S. Pat. No. 6,051,206, in U.S. Ser. No. 08/253,973; and in WO 95/00553 and WO 95/33497.
A large body of literature exists relating to clinical uses of unlabeled octreotide and lanreotide. For example, as summarized in Lamberts, et al. (1996) New England J. of Med. 334, 246-254, octreotide has been investigated for use in treating thyrotropin-secreting pituitary adenomas, nonsecretory pituitary adenomas, and corticotropin-secreting pituitary adenomas such as bronchial and thymic carcinoids, medullary thyroid carcinomas and pancreatic islet cell tumors, but not those not associated with Cushing""s disease. Lamberts, et al. discloses that in general, octreotide treatment only occasionally resulted in transient inhibition of tumor growth. Lamberts, et al. further discloses that octreotide has also been studied for use in gastrointestinal and pancreatic diseases, with variable results: for example, octreotide was not effective in treating bleeding from peptic ulcers, but was effective in controlling bleeding from esophageal varices. Lamberts, et al. describes octreotide as being ineffective in the treatment of acute pancreatitis, but efficacious in reducing fluid production by pancreatic fistulas and pseudocysts. Clinical trials of octreotide for treatment of watery diarrhea in AIDS patients were also described in Lamberts, et al. Octreotide has also been studied as an anti-angiogenic agent, as summarized in Woltering, et al. (1997) Investigational New Drugs 15, 77-86. Lanreotide has been applied to surgical wounds induced in tumor-implanted mice to study its effect on wound-induced acceleration of tumor growth, and its use has been suggested as an endocrine antisecretogogue in cytoreductive cancer treatment, in Bogden, et al. (1997) Brit. J. Cancer 75, 1021-1027.
Despite the commercial availability of octreotide, lanreotide, and pentetreotide, a large number of somatostatin analogs have been proposed for use as imaging and/or therapeutic agents to detect and/or treat cancer and other somatostatin-responsive disease states. Patel, et al., supra, discloses the need for second generation somatostatin analogs which bind more selectively, i.e., with higher affinity, to SSTRs generally and to SSTR subtypes, in particular to SSTR1, SSTR3, and SSTR4. Higher affinity analogs for SSTR2 and SSTR5 are also desirable, so that lower dosages of somatostatin analogs may be administered to obtain a clinical response.
Second generation somatostatin analogs are described, for example, in commonly assigned U.S. Pat. Nos. 5,620,675; 5,716,596; 5,783,170; 5,814,298; 5,820,845; 5,833,942; 5,843,401; 5,871,711; 5,932,189; allowed U.S. Ser. Nos. 08/592,323; 08/586,670; 08/776,160; 08/931,095; 09/039,062; 09/039,116; 09/042,224 and 09/042,315; and copending U.S. Ser. No. 08/092,355. U.S. Pat. No. 5,597,894 discloses somatostatin analogs containing additional N-terminal amino acids. U.S. Pat. Nos. 4,310,518 and 4,486,415 and EP 143 307 and EP 222 578 disclose cyclic hexapeptide somatostatin analogs which are cyclized through peptide linkages. U.S. Pat. No. 5,708,135 discloses somatostatin analogs which are cyclized through a disulfide bond between the N-terminal residue and the C-terminal residue. U.S. Pat. No. 5,776,894 discloses somatostatin peptides having a chelating group covalently linked to an N-terminal amino group. U.S. Pat. No. 5,770,687 discloses conformationally constrained backbone cyclized somatostatin analogs. U.S. Pat. No. 5,830,431 discloses radiolabeled somatostatin analogs having carboxyl termini in the carboxylic acid form. EP 714 911 discloses DOTA-conjugated octreotide analogs. WO 96/37239 discloses 188Re-labeled vapreotide. WO 97/01579 discloses cyclic hexapeptide somatostatin analogs having a chelating group attached to a side chain amino group of a designated amino acid. Even in light of the large number of second generation somatostatin analogs which has been proposed, research continues for somatostatin analogs with improved binding properties.
Radiolabeled forms of octreotide and lanreotide have been investigated for use as radiotherapeutic agents in preclinical and clinical studies. For example, Anderson, et al. (1996) J. Nucl. Med. 37, 128P-129P, discloses a study of 64Cu-labeled TETA-octreotide as a radiotherapeutic in a tumor-bearing rat model. Stolz, et al. (1996) Digestion 57 (suppl. 1) 17-21, discloses 90Y-DTPA-benzyl-acetamido-D-Phe1, Tyr3-octreotide in a tumor-bearing mouse model. Otte, et al. (1997) Eur. J. Nucl. Med. 24, 792-795, discloses use of 90Y-labeled DOTA-D-Phe1, Tyr3-octreotide to treat metastatic neuroendocrine disease in one human patient. Krenning, et al. (1994) Annals of N. Y. Acad. Sci. 733, pp. 496-506, describes radiotherapy of a patient with a neuroendocrine tumor using therapeutic doses of 111In-DTPA-D-Phe-octreotide. DeJong, et al. (1998) Int. J. Cancer 75, 406-411, discloses a preclinical comparison of {DTPA}-octreotide, {DTPA, Tyr3}-octreotide, and {DOTA, Tyr3}-octreotide labeled with 111In and 90Y. Breeman, et al. (1998) Eur. J. Nucl. Med. 25, 182-186, describes a comparison of 111In-DTPA-RC-160 (vapreotide) and 111In-DTPA-octreotide for somatostatin receptor scintigraphy in humans. WO 98/41540 discloses DOTA-lanreotide for labeling with 111In, 90Y, 86Y, 67/68Ga, 161Tb, and 153Sm. Virgolini, et al. (1998) J. Nucl. Med. 39, 1928-1936, describes biodistribution of 111In-DOTA-lanreotide in humans. Leimer, et al. (1998) J. Nucl. Med. 39, 2090-2094, describes treatment of a metastatic carcinoma in a human patient using 90Y-DOTA-lanreotide. Preliminary data indicate that when labeled with a radionuclide having sufficiently energetic emissions, octreotide and lanreotide may be effective antitumor agents for somatostatin receptor-bearing tumors.
A frequently observed phenomenon with radiolabeled peptides is retention in a non-target organ such as kidney, liver, or spleen. When short-lived, relatively low energy isotopes such as 99mTc are employed, kidney retention generally does not present a clinical problem. However, use of peptides labeled with isotopes having higher energies and/or longer half-lives, such as those proposed for use in radiotherapy, could result in an unacceptably high radiation dose to the non-target organ. Indeed, Breeman, et al., supra, concludes that the high kidney, liver, spleen, and lung background of 111In-DTPA-vapreotide rendered the compound unsuitable for scintigraphy. Retention of radiolabeled peptides in non-target organs limits the amount of radioactivity which can be administered.
Bernard, et al. (1997) J. Nucl. Med. 38, 1929-1933 discloses that significant amounts of 111In-DTPA-octreotide and 90Y-DOTA-octreotide accumulate in the renal parenchyma when administered to normal rats. Bass, et al. (1998) Bioconjugate Chem. 9, 192-200, discloses that 111In-DTPA-octreotide is retained in liver and kidney of normal and tumor-bearing rats. Virgolini, et al., supra, discloses that at 24 hours post-injection, 111In-DOTA-lanreotide is retained in human liver to about the same extent as 111In-DTPA- D-Phe1-octreotide (about 3.2% injected dose); in spleen to a smaller extent (about 1.2% injected dose for 111In-DOTA-lanreotide compared to about 3% injected dose for 111In-DTPA-D-Phel-octreotide); and in kidney to a smaller extent (about 2.5% injected dose for 111In-DOTA-lanreotide compared to about 3.7% injected dose for 111In-DTPA-D-Phe1-octreotide). Leimer, et al., supra, reports 90Y kidney doses of 1.98-2.43 mGy/MBq and spleen doses of 1.68-3.35 mGy/MBq in the patient treated with four cycles of 90Y-DOTA-lanreotide treatment.
Several approaches have been suggested to minimize the accumulation of radiolabeled peptides in non-target organs. Use of D- or L-lysine to reduce renal uptake of antibodies and/or peptides is disclosed in U.S. Pat. Nos. 5,380,513; 5,648,059; and 5,843,894. Stolz, et al. (1998) Eur. J. Nucl. Med. 25, 668-674, discusses use of kidney-specific cleavable linkers to enhance excretion of radiolabeled peptides but suggests using prior injection of L-lysine to lower the kidney radiation dose resulting from administration of 90Y-DOTA-D-Phe1, Tyr3-octreotide. Bernard, et al., supra, teaches that high doses of L-lysine may result in toxicity and suggests use of D-lysine to reduce accumulation of 111In-DTPA-octreotide and 90Y-DOTA-octreotide in kidneys. Hammond, et al. (1993) Brit. Cancer J. 67, 1437-1439, teaches infusion of a mixture of lysine and arginine to prevent kidney uptake of 111In-pentetreotide in human patients.
Although infusion of amino acids reduces kidney uptake of radiolabeled somatostatin analogs, the potential exists that the infused amino acids may themselves result in toxicity. In addition, currently there are no commercially available amino acid infusates with sufficiently high concentrations of lysine to effectively reduce kidney uptake of radiolabeled somatostatin analogs. A decrease in renal accumulation of a radiolabeled somatostatin analog would allow administration of a higher radiation dose, and thus more effective tumor ablation would be expected.
Thus a need remains for novel somatostatin analogs with improved affinity for SSTRs and improved pharmacokinetic properties.
The present inventors have designed novel SSTR pharmacophores which are structurally distinct from all previously known somatostatin analogs. The novel pharmacophores of the invention exhibit high affinity for SSTRs and consequently high tumor uptake. The inventors have also discovered novel metal ion chelators, which when covalently linked to the pharmacophores of the invention result in a decrease in kidney, spleen, gastrointestinal tract, and/or liver retention, thus enhancing the utility of pharmacophores for use with cytotoxic radioisotopes. In addition, the combination of the novel chelators of the invention with the novel pharmacophores disclosed herein results in high tumor uptake of the pharmacophore. The somatostatin analogs of the invention may be administered to treat somatostatin-responsive disease states, and in addition may be labeled with a radioisotope for use as diagnostic or therapeutic agents.
In one embodiment, the invention provides a compound comprising a somatostatin receptor-binding peptide having a formula
cyclo-B1-B2-B3-B4-C-A
wherein
B1 is Phe, Tyr, Nal, Ain, or a substituted derivative thereof;
B2 is Trp or a substituted derivative thereof;
B3 is Lys, Hly, Achxa, Amf, Aec, Apc, Aes, Aps or a substituted derivative thereof;
B4 is Thr, Ser, Val, Phe, Ile, Abu, Nle, Leu, Nva, or Aib;
C is an L-xcex1-amino acid;
A is an N-alkyl-xcex1-amino acid or an N-substituted alkyl-xcex1-amino acid, wherein A comprises a sidechain containing a sulfur atom;
wherein B1 and A are covalently linked through an amino terminus of B1 and a carboxyl terminus of A to form a cyclic peptide. In accordance with the invention, the A residue may be linked to a metal chelator through a sidechain nitrogen, carbon, sulfur, or oxygen atom. The compounds of the invention are characterized by molecular weights of less than about 10,000 daltons.
In another embodiment, the invention provides a compound comprising a somatostatin receptor-binding peptide having a formula
cyclo-B1-B2-B3-B4-C-(Nxe2x80x94CH3)Hcy(X)
wherein
B1 is Phe, Tyr, Nal, Ain, or a substituted derivative thereof;
B2 is Trp or a substituted derivative thereof;
B3 is Lys, Hly, Achxa, Amf, Aec, Apc, Aes, Aps or a substituted derivative thereof;
B4 is Thr, Ser, Val, Phe, Ile, Abu, Nle, Leu, Nva, or Aib;
C is an L-xcex1-amino acid;
X is a H or a metal chelator;
wherein B1 and (Nxe2x80x94CH3)Hcy are covalently linked through an amino terminus of B1 and a carboxyl terminus of (Nxe2x80x94CH3)Hcy to form a cyclic peptide. In accordance with the invention, X is linked to the (Nxe2x80x94CH3)Hcy residue through the Hcy sidechain sulfur atom.
In another embodiment, the invention provides a novel peptide chelator having a formula:
-xcex2-Dap.Xaa.Cys.Zaa
wherein
Xaa is an L-xcex1-amino acid, and
Zaa is an xcex1-amino acid, an xcex1-amino acid amide, an aminoethylether, a xcex2-aminol, or a peptide containing from two to ten xcex1-amino acids, said peptide having a carboxyl terminal xcex1-amino acid, xcex1-amino acid amide, aminoethylether, or xcex2-aminol.
In another embodiment, the invention provides a radiopharmaceutical comprising a compound of the invention and a radioisotope which may be a xcex3-emitter for imaging purposes, or alternatively, an xcex1-emitter or a xcex2-emitter for therapeutic purposes.
In another embodiment, the invention provides a kit comprising a sealed vial containing a predetermined quantity of a compound of the invention. The sealed vial may optionally contain a sufficient amount of a reducing agent to label the compound with 99mTc, 186Re, or 188Re.
In yet another embodiment, the invention provides a method of imaging a site within a mammalian body, comprising the steps of radiolabeling a compound of the invention with a suitable xcex3-emitting radioisotope, administering an effective diagnostic amount of the radiolabeled compound to the body, and detecting the radioisotope accumulated at the site.
The invention also provides a method of treating an animal suffering a somatostatin-responsive disease, comprising the steps of radiolabeling a compound of the invention with an xcex1-emitting or a xcex2-emitting radioisotope, and administering a therapeutically effective amount of the radiolabeled compound to the animal.
The diagnostic and therapeutic methods of the invention may be combined to treat an SSTR-bearing tumor in a mammal, wherein a first aliquot of a compound of the invention is labeled with a xcex3-emitting radioisotope and used for diagnostic imaging, and if accumulation of the xcex3-emitting radioisotope is observed, a second aliquot of the same compound or a pharmacologically similar compound is labeled with a cytotoxic radioisotope and administered to the mammal to effect tumor ablation.
The patent and scientific literature referenced herein establish the knowledge available to those with skill in the art. The issued U.S. patents and allowed applications are hereby incorporated by reference.
The novel pharmacophores of the invention bind with high affinity to SSTRs, and when covalently linked to the novel metal chelators of the invention, demonstrate high tumor uptake and, in addition, accumulate in liver, spleen, and/or kidney to a lower extent than known somatostatin analogs. The preferred compounds of the invention demonstrate improved properties such as subnanomolar affinity for SSTRs, high tumor uptake, and/or minimal kidney and/or gastrointestinal tract accumulation when radiolabeled.
The novel compounds of the invention may be used in unlabeled or labeled form to treat any somatostatin-responsive disease state. In general, somatostatin-responsive disease states are characterized by expression or over-expression of SSTRs. Exemplary somatostatin-responsive disease states include, without limitation, endocrine tumors such as those described supra, gastrointestinal tract tumors, breast carcinoma, small cell carcinoma of the lung, lymphoma, neuroblastoma, peptide hypersecretion from functional endocrine tumors of the gastroenteropancreatic axis, growth hormone hypersecretion in acromegaly, diabetic retinopathy, gut hypersecretion and increased motility in the dumping syndrome, pancreatic and gastrointestinal tract fistulas, complications following pancreatic surgery, increased portal pressure and variceal bleeding associated with esophageal varices, and the like.
As set forth above, the novel pharmacophore of the invention is embodied as a compound having the formula
cyclo-B1-B2-B3-B4-C-A
wherein
B1 is Phe, Tyr, Nal, Ain, or a substituted derivative thereof;
B2 is Trp or a substituted derivative thereof;
B3 is Lys, Hly, Achxa, Amf, Aec, Apc, Aes, Aps or a substituted derivative thereof;
B4 is Thr, Ser, Val, Phe, Ile, Abu, Nle, Leu, Nva, or Aib;
C is an L-xcex1-amino acid;
A is an N-alkyl-xcex1-amino acid or an N-substituted alkyl-xcex1-amino acid, wherein A comprises a sidechain containing a sulfur atom;
wherein B1 and A are covalently linked through an amino terminus of B1 and a carboxyl terminus of A to form a cyclic peptide. In the formula set forth above, xe2x80x9csubstitutedxe2x80x9d is intended to include such substitutions as H, amino, hydroxyl, N-alkyl wherein alkyl represents C1 to C4 alkyl, N, N-dialkyl wherein alkyl represents C1 to C4 alkyl, N-aryl, N-acyl, O-alkyl wherein alkyl represents C1 to C4 alkyl, O-aryl, O-acyl, S-alkyl wherein alkyl represents C1 to C4 alkyl, S-aryl, and the like. In accordance with the invention, the amino acids set forth above may be in the D- or L- configuration. As defined herein, xe2x80x9ccontaining a sulfur atomxe2x80x9d means that the sidechain of the A residue comprises such groups as xe2x80x94SH, xe2x80x94Sxe2x80x94, xe2x80x94Sxe2x80x94CH2COOH, xe2x80x94Sxe2x80x94CH2COOxe2x80x94, xe2x80x94Sxe2x80x94CH2xe2x80x94CH(COxe2x80x94)(NHxe2x80x94), and the like.
Preferably, B1 is L-Phe or L-Tyr; B2 is D-Trp; B3 is L-Lys; and B4 is L-Thr or L-Val, and A is an N-methyl-xcex1-amino acid. More preferably, A is selected from the group consisting of (Nxe2x80x94CH3)Cys, (Nxe2x80x94CH3)Hcy, (Nxe2x80x94CH3)Tyr, (Nxe2x80x94CH3)Tty, and (Nxe2x80x94CH3)Tyr(CH2CH2SH). Most preferably, A is (Nxe2x80x94CH3)Tyr, (Nxe2x80x94CH3)Cys, or (Nxe2x80x94CH3)Hcy. When A is (Nxe2x80x94CH3)Tyr, (Nxe2x80x94CH3)Cys, or (Nxe2x80x94CH3)Hcy, C is preferably L -methionine, L -phenylalanine, or a substituted derivative of L-phenylalanine. More preferably, A is (Nxe2x80x94CH3)Cys or (Nxe2x80x94CH3)Hcy and C is L-phenylalanine or a substituted derivative of L -phenylalanine. Most preferably, A is (Nxe2x80x94CH3)Hcy and C is L-phenylalanine. In a most preferred embodiment, the pharmacophore of the invention has the formula:
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy.
The use of the term xe2x80x9ccycloxe2x80x9d and underlining of amino acids herein is intended to indicate that the peptide is cyclized by formation of an amide linkage between the substituted or unsubstituted amine group of the first underlined amino acid and the carboxyl group of the last underlined amino acid. Use of smaller font indicates that the chemical symbol for an atom is intended rather than a one-letter amino acid code.
All naturally-occurring amino acids may be referenced herein in abbreviated form, using standard one-letter or three-letter codes (which can be found in G. Zubay, Biochemistry (2d. ed.), 1988 (MacMillen Publishing: New York) p.33). In addition, as used herein, the following amino acids and amino acid analogues are intended to be represented by the following abbreviations: Hcy is homocysteine; Hhc is homohomocysteine (3-mercaptopropylglycine); Pen is penicillamine; Aib is aminoisobutyric acid; Nal is 2-naphthylalanine; Aca is 6-aminocaproic acid; Ain is 2-aminoindan-2-carboxylic acid; Hly is homolysine; Achxa is 4-amino-cyclohexylalanine; Amf is 4-aminomethyl-phenylalanine; Aec is S-(2-aminoethyl)cysteine; Apc is S-(3-aminopropyl) cysteine; Aes is O-(2-aminoethyl)serine; Aps is O-(3-aminopropyl)serine; Abu is 2-aminobutyric acid; Nva is norvaline; FD is D-phenylalanine; WD is D-tryptophan; YD is D-tyrosine; Cpa is L-(4-chlorophenyl) alanine; Thp is 4-amino-tetrahydrothiopyran-4-carboxylic acid; D-Nal is D-2-naphthylalanine; Dpg is dipropylglycine; Nle is norleucine; (Nxe2x80x94CH3)Cys is N-methyl-cysteine; (Nxe2x80x94CH3)Hcy is N-methyl-homocysteine; (Nxe2x80x94CH3)Tyr is N-methyl-tyrosine; (Nxe2x80x94CH3)Tty is N-methyl-thiotyrosine (i.e., N-methyl-4-mercaptophenylalanine); (Nxe2x80x94CH3)Tyr(CH2 CH2 SH) is N-methyl-O-2-mercaptoethyl tyrosine; Thr(OH) is threoninol residue (wherein the carboxyl group of the amino acid is reduced to a primary alcohol, incorporated into the peptide using the procedure of Neugebauer et al. (1990, Peptides: Proceedings of the 11th American Peptide Symposium, pp. 1020-21); Ser(ol) is. serinol; Asp(ol) is aspartinol; Glu(ol) is glutarinol; Gln(ol) is glutaminol; Asn(ol) is asparaginol; Phe(4-F) is 4-fluoro-phenylalanine; Phe(4-NH2) is 4-amino-phenyalanine; xcex5-Lys represents a covalent linkage via the xcex5-amino group on the sidechain of a lysine residue; xcex4-Orn represents an ornithine residue in which the xcex4-amino group is covalently linked to the carboxyl group of the adjacent amino acid to form a peptide bond; xcex3-Dab represents a 2,4-diaminobutyric acid residue in which the xcex3-amino group is covalently linked to the carboxyl group of the adjacent amino acid to form a peptide bond; xcex2-Dap represents a 2,3-diaminopropionic acid residue in which the xcex2-amino group is covalently linked to the carboxyl group of the adjacent amino acid to form a peptide bond. When a combination of one-letter codes is used with the abbreviations set forth above, the abbreviation is set off by periods.
In accordance with the present invention, a xe2x80x9csubstituted derivativexe2x80x9d of an amino acid includes such substitutions as amino, hydroxyl, N-alkyl wherein alkyl represents C1 to C4 alkyl, N-aryl, N-acyl, O-alkyl wherein alkyl represents C1 to C4 alkyl, O-aryl, O-acyl, S-alkyl wherein alkyl represents C1 to C4 alkyl, S-aryl. When applied to amino acids that contain a sidechain aromatic ring, the term also encompasses o-, m-, and p-substitutions including but not limited to o-amino, m-amino, p-amino, amino, o-hydroxyl, m-hydroxyl, p-hydroxyl, and the like.
The invention is also embodied as a compound having the formula:
cyclo-B1-B2-B3-B4-C(Nxe2x80x94CH3)Hcy(X)
wherein B1 is Phe, Tyr, Nal, Ain, or a substituted derivative thereof;
B2 is Trp or a substituted derivative thereof;
B3 is Lys, Hly, Achxa, Amf, Aec, Apc, Aes, Aps or a substituted derivative thereof;
B4 is Thr, Ser, Val, Phe, lie, Abu, Nle, Leu, Nva, or Aib;
C is L-methionine, L-phenylalanine, or a substituted derivative of L-phenylalanine;
X is H or a metal chelator;
wherein B1 and (Nxe2x80x94CH3)Hcy are covalently linked through an amino terminus of B1 and a carboxyl terminus of (Nxe2x80x94CH3)Hcy to form a cyclic peptide. In accordance with the invention, X is linked to the (Nxe2x80x94CH3)Hcy residue through the Hcy sidechain sulfur atom. The amino acids in the formula set forth above may be in the D- or L-configuration. In this embodiment, B1 is preferably L-Phe or L-Tyr; B2 is preferably D-Trp; B3 is preferably L-Lys; and B4 is preferably L-Thr or L-Val.
In accordance with the invention, any metal chelator may be employed as substituent X. For example, the compounds of the invention may comprise a metal ion chelator having a formula:
C(pgp)s-(aa)-C(pgp)s
where (pgp)s is hydrogen or a thiol protecting group and (aa) is any xcex1- or xcex2-amino acid not comprising a thiol group. In a preferred embodiment, the amino acid is glycine. Methods for making such a metal ion chelator are set forth in U.S. Pat. Nos. 5,654,272; 5,681,541; 5,788,960; and 5,811,394.
Alternatively, the compound of the invention may comprise a metal ion chelator capable of forming an electrically neutral complex with the metal ion, as set forth in U.S. Pat. Nos. 5,720,934; 5,776,428; 5,780,007; 5,922,303, 5,965,107, 6,086,849 and 6,093,383. Such chelators include, but are not limited to 
wherein
Xxe2x80x2=H or a protecting group;
(amino acid)=any amino acid; 
wherein
Xxe2x80x2=H or a protecting group;
(amino acid)=any amino acid; 
wherein each R is independently H, CH3 or C2H5; each (pgp)s is independently a thiol protecting group or H; m, n and p are independently 2 or 3; A is linear C1-C8 alkyl, substituted linear C1-C8 alkyl, cyclic C3-C8 alkyl, substituted cyclic C3-C8 alkyl, aryl, substituted aryl, or a combination thereof; and 
wherein each R is independently H, CH3 or C2H5; m, n and p are independently 2 or 3; A is linear C1-C8 alkyl, substituted linear C1-C8 alkyl, cyclic C3-C8 alkyl, substituted cyclic C3-C8 alkyl, aryl, substituted aryl, or a combination thereof; V is H or CO-peptide; Rxe2x80x2 is H or peptide; provided that when V is H, Rxe2x80x2 is peptide and when Rxe2x80x2 is H, V is COxe2x80x94 pharmacophore. In accordance with the invention, the substituted derivatives in the bisamine, bisthiol formulae are defined as set forth above.
Alternatively, the compound of the invention may comprise a metal ion chelator having a formula selected from the group consisting of:
diethylenetriaminepentaacetic acid (DTPA);
a derivative of DTPA having a formula
(HOOCCH2)2N(CR2)(CR2)N(CH2COOH)(CR2)(CR2)N(CH2COOH)2;
where each R is independently H, C1 to C4 alkyl, or aryl and one R is covalently linked to a bivalent linker;
ethylenediaminetetraacetic acid (EDTA);
a derivative of EDTA having a formula
(HOOCCH2)2N(CR2)(CR2)N(CH2COOH)2;
where each R is independently H, C1 to C4 alkyl, or aryl and one R is covalently linked to a bivalent linker;
1,4,7,10-tetraazacyclododecanetetraacetic acid and derivatives thereof such as those set forth in U.S. Pat. Nos. 4, 923,985; 5,053,503; 5,428,154; WO 94/27977, EP 512 661; and the like;
a metal ion chelator having a formula: 
where n is an integer that is 2 or 3 and where each R is independently H, C1 to C4 alkyl, or aryl and one R is covalently linked to the peptide; and desferrioxamine.
In addition to the chelators set forth above, the somatostatin receptor-binding peptides of the invention may be covalently linked to radiometal binding ligand such as a hydrazino nicotinamide moiety and radiolabeled using appropriate co-ligands. Such ligands and co-ligands are disclosed, for example, in U.S. Pat. Nos. 5,300,278; 5,350,837; 5,589,576; 5,679,778; and 5,879,659, and in Spies, et al. Technetium, Rhenium and Other Metals in Chemistry and Nuclear Medicine, Nicolini, et al., eds., SGEditoriali (Padua, 1999) pp. 101-108 and 687-690.
More preferably, the compounds of the invention comprise a monoamine, diamide, single thiol containing metal ion chelator such as those set forth in commonly assigned copending U.S. Ser. No. 08/253,973. Exemplary of such metal ion chelators are chelators having the formulae: 
wherein n, m and p are each integers that are independently 0 or 1; each Rxe2x80x2 is independently H, lower alkyl, C2-C4 hydroxyalkyl, or C2-C4 alkoxyalkyl, and each R is independently H or Rxe2x80x3, where Rxe2x80x3 is a substituted C1-C8 alkyl not comprising a thiol group, a unsubstituted C1-C8 alkyl, an unsubstituted phenyl, or a substituted phenyl not comprising a thiol group, and one R or Rxe2x80x2 is L2, where L2 is a bivalent linker moiety linking the metal chelator to the peptide and wherein when one Rxe2x80x2 is L2, NRxe2x80x22 is an amine. In this embodiment, L2 may be a C1-C6 linear alkyl group, a branched chain alkyl group, a cyclic alkyl group, a carboxylic ester, a carboxamide, a sulfonamide, an ether, a thioether, an amine, an alkene, an alkyne, a 1,2-linked, optionally substituted, benzene ring, a 1,3-linked, optionally substituted, benzene ring, a 1,4-linked, optionally substituted, benzene ring, or an amino acid, or a combination thereof In this embodiment, Rxe2x80x3 may be a C1-C6 linear alkyl group; a branched alkyl group; a cyclic alkyl group; a xe2x80x94CqOCrxe2x80x94, xe2x80x94CqNHCrxe2x80x94 or xe2x80x94CqSCrxe2x80x94 group, where q and r are integers each independently 1 to 5 wherein the sum of q+r is not greater than 6; (C1-C6) alkyl-X, where X is a hydroxyl group, a substituted amine, a guanidine, an amidine, a substituted thiol group, or a carboxylic acid, ester, phosphate, or sulfate group; a phenyl group or a phenyl group substituted with a halogen, a hydroxyl group, a substituted amine, a guanidine group, an amidine group, a substituted thiol, an ether, a phosphate, a sulfate; an indole group; a C1-C6 heterocyclic group containing 1 to 3 nitrogen, oxygen or sulfur atoms, or a combination thereof. In accordance with the invention, the substituted derivatives in the monoamine, diamide, thiol-containing chelator formulae are defined as set forth above.
Most preferably, the compounds of the invention comprise a metal ion chelator comprising a single thiol-containing group of formula:
Axe2x80x94CZ(B)xe2x80x94{C(RaRb)}nxe2x80x94X
wherein A is H, HOOCxe2x80x94, H2NOCxe2x80x94, xe2x80x94NHOCxe2x80x94, xe2x80x94OOCxe2x80x94, Re2NOCxe2x80x94, or Rd; B is H, SH, xe2x80x94NHRc, N(Rc)xe2x80x94 or Rd; Z is H or Rd; X is SH, xe2x80x94NHRc, xe2x80x94N(Rc)xe2x80x94 or Rd; Ra, Rb, Rc and Rd are independently H, straight chain C1-C8 alkyl, branched chain C1-C8 alkyl, or cyclic C3-C8 alkyl; n is 0, 1 or 2; Re is C1-C4 alkyl, an amino acid, or a peptide comprising 2 to about 10 amino acids; and: (1) where B is xe2x80x94NHRc or xe2x80x94N(Rc)xe2x80x94, X is SH and n is 1 or 2; (2) where X is xe2x80x94NHRc or xe2x80x94N(Rc)xe2x80x94, B is SH and n is 1 or 2; (3) where B is H or Rd, A is HOOCxe2x80x94, H2NOCxe2x80x94, xe2x80x94NHOCxe2x80x94, or xe2x80x94OOCxe2x80x94, X is SH and n is 0 or 1; (4) where A is H or Rd, then where B is SH, X is xe2x80x94NHRc or xe2x80x94N(Rc)xe2x80x94 and where X is SH, B is xe2x80x94NHRc or xe2x80x94N(Rc) and n is 1 or 2; (5) where X is H or Rd, A is HOOCxe2x80x94, H2NOCxe2x80x94, xe2x80x94NHOCxe2x80x94, or xe2x80x94OOCxe2x80x94 and B is SH; (6) where Z is methyl, X is methyl, A is HOOCxe2x80x94, H2NOCxe2x80x94, xe2x80x94NHOCxe2x80x94, or xe2x80x94OOCxe2x80x94 and B is SH and n is 0; and (7) where B is SH, X is not SH and where X is SH, B is not SH.
In accordance with the invention, a metal ion chelator comprising a single thiol-containing group may have the formula:
R1xe2x80x94CO-(amino acid)1-(amino acid)2-Z1
wherein (amino acid)1 and (amino acid)2 are each independently any primary xcex1- or xcex2-amino acid that does not comprise a thiol group, Z1 is selected from the group consisting of cysteine, homocysteine, isocysteine, penicillamine, 2-mercaptoethylamine, 2-mercaptopropylamine, 2-mercapto-2-methylpropylamine, and 3-mercaptopropylamine, and R1 is lower (C1-C4) alkyl, or R1xe2x80x94CO is an amino acid, a peptide, or (aa)-peptide; wherein when Z1 is cysteine, homocysteine, isocysteine or penicillamine, Z1 comprises a carbonyl group covalently linked to a hydroxyl group, a NR3R4 group, wherein each of R3 and R4 are independently H, a bond, lower (C1-C4) alkyl, an amino acid or a peptide comprising from 2 to 10 amino acids.
Alternatively, a metal ion chelator comprising a single thiol-containing group may have the formula:
Y-(amino acid)2-(amino acid)1-NHR2
wherein (amino acid)1 and (amino acid)2 are each independently any primary xcex1- or xcex2-amino acid that does not comprise a thiol group Y is selected from the group consisting of cysteine, homocysteine, isocysteine, penicillamine, 2-mercaptoacetate, 2-mercaptopropionate, 2-mercapto-2-methylpropionate, 3-mercaptopropionate, and R2 is H, a bond, lower (C1-C4) alkyl, and NHR2 is an amino acid, a peptide, or (aa)-peptide; wherein when Y is cysteine, homocysteine, isocysteine or penicillamine, Y comprises an amino group covalently linked to xe2x80x94H, an amino acid, a peptide, or (aa)-peptide.
For example, suitable metal ion chelators may have any of the following formulae:
(amino acid)1-(amino acid)2-cysteine-,
(amino acid)1-(amino acid)2-isocysteine-,
(amino acid)1-(amino acid)2-homocysteine-,
(amino acid)1-(amino acid)2-penicillamine-,
(amino acid)1-(amino acid)2-2-mercaptoethylamine-,
(amino acid)1-(amino acid)2-2-mercaptopropylamine-,
(amino acid)1-(amino acid)2-2-mercapto-2-methylpropylamine-,
(amino acid)1-(amino acid)2-3-mercaptopropylamine-,
wherein the chelator is attached to either a peptide or a linker group via a covalent bond with the carboxyl terminus of the chelator or a side chain on one of the amino acid groups.
Other suitable metal ion chelators include those selected from the group consisting of:
-cysteine-(amino acid)-(xcex1,xcex2- or xcex2,xcex3-diamino acid);
-isocysteine-(amino acid)-(xcex1,xcex2- or xcex2,xcex3-diamino acid);
-homocysteine-(amino acid)-(xcex1,xcex2- or xcex2,xcex3-diamino acid);
-penicillamine-(amino acid)-(xcex1,xcex2- or xcex2,xcex3-diamino acid);
2-mercaptoacetic acid-(amino acid)-(xcex1,xcex2- or xcex2,xcex3-diamino acid);
2- or 3-mercaptopropionic acid-(amino acid)-(xcex1,xcex2- or xcex2,xcex3-diamino acid);
2-mercapto-2-methylpropionic acid-(amino acid)-(xcex1,xcex2- or xcex2,xcex3-diamino acid);
wherein the metal ion chelator is attached to either a peptide or a linker group via a covalent bond with the amino terminus of the chelator or a side chain on one of the amino acid groups.
Any naturally occurring, modified, substituted, or altered xcex1- or xcex2-amino acid not containing a thiol group may be used in the single-thiol chelators of the invention. As used herein, the term xe2x80x9cmodified, substituted, or altered xcex1- or xcex3-amino acidxe2x80x9d includes, without limitation, any of the amino acids identified above. Preferably, the xcex1- or xcex2-amino acid does not comprise more than sixteen carbon atoms.
The most preferred novel chelators of the invention have the formula:
xcex2-Dap.Xaa.Cys.Zaa
wherein
Xaa is an L-xcex1-amino acid
Zaa is an xcex1-amino acid, an xcex1-amino acid amide, an aminoethylether, a xcex2-aminol, or a peptide containing from two to ten xcex1-amino acids, said peptide having a carboxyl terminal xcex1-amino acid, xcex1-amino acid amide, aminoethylether, or xcex2-aminol.
Homologs of xcex2-Dap may also be employed in the novel chelators of the invention.
Suitable L-xcex1-amino acids for substitution as Xaa in the novel chelator of the invention include naturally occurring amino acids such as asparagine, glutamine, threonine, serine, arginine, histidine, lysine, ornithine, phenylalanine, tyrosine, and, in addition, synthetic amino acids containing hydrophilic substituents. Exemplary synthetic amino acids include, without limitation, diaminopropionic acid; diaminobutyric acid; substituted tyrosines such as halotyrosine; hydroxyltyrosine; aminotyrosine; substituted phenylalanines such as o-, m- or p-halophenylalanine; o-, m- or p-aminophenylalanine, wherein the amino substitutent may be a primary, secondary, or tertiary amine; o-, m- or p-hydroxylphenylalanine; o-, m- or p-O-alkylphenyalanine wherein alkyl represents C1 to C4 alkyl; o-, m- or p-O-acylphenylalanine; o-, m- or p-S-alkylphenylalanine wherein alkyl represents C1 to C4 alkyl; and the like
In a preferred embodiment, Xaa is an L-xcex1-amino acid such as serine, diaminobutyric acid, arginine, histidine, tyrosine, or a substituted phenylalanine. More preferably, Xaa is an aromatic an L-xcex1-amino acid such as tyrosine, a substituted tyrosine residue such as iodotyrosine, bromotyrosine, chlorotyrosine, O-alkyl-tyrosine where alkyl represents C1 to C8 alkyl, hydroxyltyrosine, aminotyrosine, and the like, or a substituted phenylalanine residue. Most preferably, Xaa is a substituted phenylalanine residue wherein the substitutions include halogen, amino, hydroxyl, NH-alkyl wherein alkyl represents C1 to C4 alkyl, NH-acyl, O-alkyl wherein alkyl represents C1 to C4 alkyl, O-acyl, S-alkyl wherein alkyl represents C1 to C4 alkyl, SO-alkyl and SO2-alkyl wherein alkyl represents C1 to C4 alkyl, SO3H, CO2H, CO2-alkyl wherein alkyl represents C1 to C4 alkyl, CONH-alkyl wherein alkyl represents C1 to C4 alkyl. Specific embodiments of such substituted phenylalanine residues include 4-fluorophenylalanine, 4-chlorophenylalanine, 4-bromophenylalanine, 4-iodophenylalanine, 4-nitrophenylalanine, 4-aminophenylalanine, N4-R-4-aminophenylalanine, N4-R, N4-Rxe2x80x2-4-aminophenylalanine, or 3-Rxe2x80x2-4-aminophenylalanine where R is C1 to C4 alkyl and Rxe2x80x2 is selected from the group consisting of H, C1 to C4alkyl amino, hydroxyl, NH-alkyl wherein alkyl represents C1 to C4 alkyl, NH-acyl, O-alkyl wherein alkyl represents C1 to C4 alkyl, O-acyl, S-alkyl wherein alkyl represents C1 to C4 alkyl, SO-alkyl and SO2-alkyl wherein alkyl represents C1 to C4 alkyl, SO3H, CO2H, CO2-alkyl wherein alkyl represents C1 to C4 alkyl, CONH-alkyl wherein alkyl represents C1 to C4 alkyl. The structures of exemplary most preferred substituted phenylalanine residues for use in the chelators of the invention are set forth below. 
where R and Rxe2x80x2 are each independently H, a straight chain C1 to C4 alkyl group, a branched chain C1 to C4 alkyl group, or an aryl group. In accordance with the invention, the carboxyl terminal amino acid of the chelators of the invention may be in carboxylic acid form or in amidated form, or alternatively, in the form of a xcex2-aminol.
The novel chelators of the invention have the properties of increasing the tumor uptake of the pharmacophore for SSTRs and enhancing kidney clearance of the radiolabeled peptide. Consequently, the novel chelators of the invention minimize retention of radiolabeled peptide in non-target tissues.
Specific embodiments of the compounds of the invention include the following peptides.
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-xcex2-Dap-Tyr-Cys-Thr(ol))
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-xcex2-Dap-Phe(4-F )-Cys-Thr(ol))
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-xcex2-Dap-Phe(4-NH2)-Cys-Thr-Ser)
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-xcex2-Dap-Dab-Cys-Thr)
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-xcex2-Dap-Phe(4-NH2)-Cys-Thr)
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-xcex2-Dap-Phe(4-NH2)-Cys-Thr(ol))
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-xcex2-Dap-His-Cys-Thr(ol))
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-xcex2-Dap-Arg-Cys-Thr(ol))
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-xcex2-Dap-Gly-Cys-Lys-NH2)
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-xcex2-Dap-Ser-Cys-Thr(ol))
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-xcex2-Dap-Dab-Cys-Thr(ol))
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-xcex2-Dap-Gly-Cys-Thr(ol))
cyclo-Tyr-D-Try-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-xcex2-Dap-Dab-Cys-Ser(ol))
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-Gly-Gly-Cys-Lys-NH2)
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-Gly-Gly-Cys-Arg-NH2)
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-Ser-Ser-Cys-Lys-NH2)
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-Ser-Ser-Cys-Arg-NH2)
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-Ser-Ser-Cys-Lys-Thr(ol))
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-Ser-Ser-Cys-Dap-NH2)
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-Ser-Ser-Cys-NH(CH2 CH2 O)2 CH2 CH2 NH2)
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-xcex2-Dap-Ser-Cys-Thr-NH(CH2 CH2 O)2 CH2 CH2 NH2)
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-Gly-Lys-Cys-NH2)
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-Ser-Lys-Cys-NH2)
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-Lys-Gly-Cys-NH2)
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-Ser-Dab-Cys-Ser(ol))
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-Ser-Dap-Cys-NH2)
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-Gly-Gly-Cys-His-NH2)
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-Gly-Gly-Cys-Phe(4 NH2)-NH2)
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-xcex2-Dap-Orn-Cys-Thr(ol))
cyclo-Tyr-D-Trp-Les-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-xcex2-Dap-Dap-Cys-Thr(ol))
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-xcex2-Dap-Lys-Cys-Thr(ol))
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-Ser-Ser-Cys-NHCH2 CH2 OCH2 CH2 NH2)
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-xcex2-Dap-Lys-Cys-NH2)
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-xcex4-Orn-Gly-Cys-NH2)
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-Thr-Gly-Gly-Cys-NH2)
Preferred compounds of the invention exhibit high affinity for SSTRs, as indicated in Example 5. In accordance with the invention, high affinity for SSTRs is defined as less than about 25 nanomolar affinity for SSTRs as measured in a SSTR binding affinity assay such as the assay set forth in Example 5. More preferred compounds of the invention are characterized by high affinity for SSTRs, as indicated in Example 5, and by high tumor uptake, as indicated in Example 6. As defined herein, high tumor uptake means a percent injected dose (% ID) of 99mTc per gram tumor (% ID/g), in the nude mouse/rat AR42J xenograft model described in Example 6, of greater than about 4. Most preferred embodiments of the compounds of the invention have a tumor uptake greater than about 15% ID/g and a kidney uptake less than about 7% ID/g to about 9% ID/g; or alternatively a tumor uptake greater than about 10% ID/g, a kidney uptake less than about 7% ID/g to about 9% ID/g, and a gastrointestinal tract uptake less than about 5% ID/g to about 7% ID/g. Exemplary most preferred compounds of the invention include:
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-xcex2-Dap-Tyr-Cys-Thr(ol))
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-xcex2-Dap-Phe(4-F)-Cys-Thr(ol))
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-xcex2-Dap-Phe(4-NH2)-Cys-Thr-Ser)
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-xcex2-Dap-Phe(4-NH2)-Cys-Thr(ol))
cyclo-Tyr-D-Trp-Lys-Thr-Phe-(Nxe2x80x94CH3)Hcy(CH2 CO-xcex2-Dap-Dab-Cys-Thr(ol))
Methods for making the compounds of the invention, and in particular those comprising the metal ion chelators of the most preferred embodiment are set forth in U.S. Pat. Nos. 5,443,815; 5,807,537; 5,814,297; 5,866,097, 5,997,844 and 6,074,627; and in U.S. Ser. Nos. 08/236,402; 08/253,973. As disclosed therein, the compounds of the invention may be made using a commercially available peptide synthesizer. Example 1 sets forth an exemplary method of making an embodiment of the pharmacophore of the present invention. Other embodiments of the novel pharmacophore are made using similar methods. Example 2 sets forth an exemplary method of making an embodiment of the novel chelator of the invention.
In accordance with the invention, the A residue may be linked to a metal chelator through a sidechain nitrogen, sulfur, or oxygen atom of the A residue. Linkage may be direct or through intervening atoms or amino acid residues. In a preferred embodiment, the linkage is through xe2x80x94CH2COxe2x80x94. Such linkages may be accomplished via the alkylation of these atoms with moieties containing reactive electrophiles such as alkyl halides. These atoms may also be reacted with chelators containing isocyanates, isothiocyanates, or activated carboxylic esters. An appropriately protected A residue may also be linked to a metal chelator through a sidechain carbon by forming a Wittig or Emmons-Homer reagent on the sidechain and reacting this with an aldehydo functionality on an appropriately protected chelator. The resulting double bond linkage can be left as is or subsequently reduced to yield saturated hydrocarbon linkage. Example 3 sets forth an exemplary method of attaching a chelator through a sidechain sulfur atom of an (Nxe2x80x94CH3)Hcy residue of the pharmacophore.
Those of skill will recognize that most metal ions may be chelated to the above-mentioned metal ion chelators and ligand/coligand radiometal binding moieties. Any metal ion capable of generating a signal may be chelated to the pharmacophore of the invention, thus forming a metal ion complex with the compound of the invention. Suitable metal ions include radioactive metal ions, fluorescent metal ions, paramagnetic metal ions, heavy metals, rare earth ions suitable for use in computerized tomography, and the like. Radioactive metal ions or radionuclides are preferred. More preferably, xcex3-emitting radionuclides such as 67Cu, 67Ga, 111In, and 99mTc; xcex2-emitting radionuclides such as 90y, 186Re, or 166Ho; xcex2/xcex3 emitting radionuclides such as 67Cu, 47Sc, 153Sm, or 188Re; positron-emitting radionuclides such as 68Ga, 94mTc, 64Cu, or xcex1-emitting radionuclides such as 213Bi, 212Bi, 225Ac, 223Ra are used in the methods of the invention. Most preferably, 99mTc, 188Re and/or 186Re are used in the methods of the invention.
Those of skill will recognize that the pharmacophores and compounds of the invention may be labeled with radioisotopes of halogens, such as 125I 131I, 123I, 18F, and 211At, through a tyrosine residue which may be added to the pharmacophore or to the compound using known methods, or using other known methods such as Bolton-Hunter reagent, chloramine T, and the like.
The compounds of the invention may also be complexed with non-radioactive metals, such as rhenium, using methods similar to those set forth below. Table 4 indicates that compounds of the invention effectively inhibit binding of 125I-Tyr11-somatostatin-14 when complexed with rhenium.
Complexes of the invention may be formed using known methods. For example, a salt of 98mTc pertechnetate, 188Re perrhenate, or 186Re perrhenate may be reacted with the compound in the presence of a reducing agent such as dithionite ion, stannous ion, or ferrous ion. In this method, the most preferred reducing agent is stannous chloride. Alternatively, complexes and chelates may be formed by ligand exchange, wherein the compound of the invention is reacted with a pre-formed labile complex of 99mTc, 188Re, or 186Re and another compound known as a transfer ligand. In this process, any transfer ligand may be used, for example, tartrate, citrate, gluconate, glucoheptonate, mannitol, and the like. Exemplary methods for labeling the compounds of the invention with 99mTc and 188Re are set forth in Example 4. In general, an appropriate quantity of a reagent of the invention is introduced into a vial containing a reducing agent, such as stannous chloride, in an amount sufficient to label the reagent with 99mTc, 188Re, or 186Re. Generally, more reducing agent is required to effect labeling with 188Re or 186Re than is required to effect labeling with 99mTc.
The compounds of the invention may be supplied in the form of a pyrogen-free, parenterally acceptable pharmaceutical composition, which may be an aqueous solution or a lyophilizate for reconstitution. The preparation of such pharmaceutical compositions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. The pharmaceutical composition of the invention may include pharmaceutically acceptable diluents, such as, for example, Sodium Chloride Injection and Ringer""s Injection. For administration to humans, the composition may be administered in autologous serum or plasma. Supplementary active compounds may also be co-administered with the somatostatin analog, in accordance with the invention.
The pharmaceutical compositions of the invention may optionally contain a stabilizer such as gentisic acid as set forth in U.S. Pat. Nos. 4,232,000; 4,233,284; 4,497,744; 5,384,113, and/or ascorbic acid as disclosed in U.S. Pat. Nos. 5,393,512 and 5,011,676, in WO 97/28181 and in WO 98/33531. Alternatively, hydroquinone stabilizers such as those disclosed in U.S. Pat. No. 4,229,427 may be added to the complexes of the invention. Other compounds such as reductic acid, erythorbic acid, p-aminobenzoic acid, 4-hydroxybenzoic acid, nicotinic acid, nicotinamide, 2,5-dihydroxy-1,4-benzenedisulfonic acid, tartaric acid, inositol, and the like, may also be added to stabilize the complexes of the invention.
The invention is also embodied in a kit for preparing radiometal-labeled reagents for use as radiopharmaceuticals. The kit of the invention comprises a sealed vial containing a predetermined quantity of the compound of the invention, and optionally, when the radiometal is 99mTc, 188Re, or 186Re, a reducing agent. Stabilizers such as gentisic acid and/or ascorbic acid or any of the stabilizers described above may also be included in kits intended for 188Re or 186Re radiolabeling. An appropriate amount of a transfer ligand as described above (such as tartrate, citrate, gluconate, glucoheptanate or mannitol, for example) can also be included in the kit. The kit may also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, buffers, preservatives, additional vials, and the like. The kit may also contain instructions for radiolabeling. The components of the kit may be in liquid, frozen or dry form. In a preferred embodiment, kit components are provided in lyophilized form.
The kit of the invention may also be embodied in a form suitable for diagnostic imaging or as a therapeutic agent using a radioisotope of a halogen, including 18F, 211At, 125I and 131I, and preferably 123I. In this embodiment, the kit comprises a sealed vial containing a predetermined quantity of a compound of the invention which may be modified to a form capable of being radiolabeled with an iodine isotope, using known methods as described above. Dose, sites and routes of administration, formulations and administered specific radioactivity using the kit of this embodiment are as described herein for technetium and rhenium-labeled reagents for scintigraphic and therapeutic uses.
The compounds of the invention may be used in radiolabeled or unlabeled form to diagnose or treat any somatostatin-responsive disease state. The compounds of the invention are particularly useful for diagnosis and/or treatment of tumors such as neuroendocrine tumors, for example, pituitary adenomas, pheochromocytomas, paragangliomas, medullary thyroid carcinomas, small cell and non small cell lung cancers, astrocytomas, melanomas, meningiomas, breast tumors, malignant lymphomas, renal cell carcinomas, prostate tumors, and the like. The compounds of the invention may also be used to diagnose or to treat conditions in which angiogenesis and concomitant upregulation of SSTRs occurs, as described in Woltering, et al., supra. As set forth in commonly assigned allowed U.S. Ser. No. 08/976,995, the compounds of the invention are useful in imaging and treating conditions of high cellular proliferation in the cardiovascular system. Such conditions include, for example, atherosclerosis and cellular proliferation occurring in arteries after invasive procedures such as angioplasty.
Preferably, radiolabeled complexes of the compounds of the invention are used for such diagnoses and treatments. Radiolabeled embodiments of the compounds of the invention may be used in radioisotope guided surgery, as described in WO 93/18797 and in Woltering, et al. (1994) Surgery 116, 1139-1147. In a preferred embodiment, a complex of a xcex3-emitting radionuclide such as 99mTc and a compound of the invention is used to diagnose an SSTR-expressing tumor, and subsequently, a complex of xcex2-emitting radionuclide such as 188Re or 186Re with the compound is used to treat the tumor.
For diagnostic purposes, an effective diagnostic amount of the diagnostic or radiodiagnostic agent of the invention is administered, preferably intravenously. An effective diagnostic amount is defined as the amount of diagnostic or radiodiagnostic agent necessary to effect localization and detection of the label in vivo using conventional methodologies such as magnetic resonance, computerized tomography, gamma scintigraphy, SPECT, PET, and the like.
For diagnosis using scintigraphic imaging, preferably, 99mTc-labeled compounds of the invention are administered in a single unit injectable dose. The 99mTc-labeled compounds provided by the invention may be administered intravenously in any conventional medium for intravenous injection such as an aqueous saline medium, or in blood plasma medium. Generally, the unit dose to be administered has a radioactivity of about 0.01 mCi to about 100 mCi, preferably 1 mCi to 50 mCi. The solution to be injected at unit dosage is from about 0.01 mnL to about 10 mL. After intravenous administration, imaging in vivo can take place in a matter of a few minutes. However, imaging can take place, if desired, hours or even longer after the radiolabeled compound is injected into a patient. In most instances, a sufficient amount of the administered dose will accumulate in the area to be imaged within about 0.1 of an hour to permit the taking of scintiphotos. Any conventional method of scintigraphic imaging for diagnostic purposes can be utilized in accordance with this invention.
When the radiolabeled compounds of the invention are used for therapeutic purposes, they are radiolabeled with a therapeutically effective amount of a cytotoxic radioisotope, preferably 188Re. In accordance with the invention, a therapeutically effective amount of a cytotoxic radioisotope means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, i.e., a reduction in the incidence or severity of symptoms attributed to the somatostatin-responsive disease state, as compared to that expected for a comparable group of patients not receiving the radiotherapeutic agent of the invention. When applied to an individual active ingredient administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially, or simultaneously. For the purposes of this invention, radiotherapy encompasses any therapeutic effect ranging from pain palliation to tumor ablation or remission of symptoms associated with the particular somatostatin-responsive disease being treated.
When used for radiotherapy, a complex of the compound of the invention and a cytotoxic radioisotope is administered to a mammal, including a human patient, in need of treatment for a somatostatin-responsive disease. In the radiotherapeutic method of the invention, an amount of cytotoxic radioisotope from about 5 mCi to about 200 mCi may be administered via any suitable clinical route, preferably by intravenous injection or by intratumoral injection. The radiotherapeutic complex of the invention may optionally be administered in combination with a chemotherapeutic drug such as tamoxifen, cisplatin, taxol, anti-angiogenic compounds, and the like.
When unlabeled compound is used for therapy of a somatostatin-responsive disease state, administration of the compound is preferably parenteral, and more preferably intravenous. The amount of unlabeled compound administered for therapy of a somatostatin-responsive disease will depend upon the nature and severity of the condition being treated, and upon the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of compound with which to treat each individual patient. Initially, the attending physician will administer low doses of the compound and observe the patient""s response. Larger doses of the compound may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the dosage of unlabeled compound administered in the therapeutic method of the invention should be in the range of about 0.1 xcexcg to about 100 mg compound per kg body weight. More preferably, the dosage of unlabeled compound administered in the therapeutic method of the invention is in the range of about 0.1 xcexcg to about 100 xcexcg compound per kg body weight. The unlabeled compound of the invention may also optionally be administered in combination with a chemotherapeutic drug.
The duration of therapy, whether with a radiopharmaceutical comprising a compound of the invention or with an unlabeled compound of the invention, will vary, depending on the severity of the disease being treated and the condition and idiosyncratic response of each individual patient. It is contemplated that the duration of each administration of the radiopharmaceutical of the invention will be in the range of about one to about 120 minutes of continuous intravenous administration. It is contemplated that the duration of each administration of the unlabeled compound of the invention will be in the range of about one to about 120 minutes of continuous intravenous administration. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy using the labeled or unlabeled compounds of the invention, whether administered alone or in combination with other drugs.